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Biol. Bull. 213: 172-186. (October 2007)
© 2007 Marine Biological Laboratory

Localization of Glutamate-like Immunoreactive Neurons in the Central and Peripheral Nervous System of the Adult and Developing Pond Snail, Lymnaea stagnalis

Dai Hatakeyama1,*, Hitoshi Aonuma2, Etsuro Ito1,3 and Károly Elekes4,{dagger}

1 Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan
2 Laboratory of Neuro-Cybernetics, Research Institute for Electronic Science, Hokkaido University, Sapporo 060-0812, Japan
3 Laboratory of Functional Biology, Faculty of Pharmaceutical Sciences at Kagawa Campus, Tokushima Bunri University, Sanuki 769-2193, Japan
4 Department of Experimental Zoology, Balaton Limnological Research Institute, Hungarian Academy of Sciences, H-8237 Tihany, Hungary

{dagger} To whom correspondence should be addressed. E-mail: elekes{at}tres.blki.hu


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
We investigated the distribution and projection patterns of central and peripheral glutamate-like immunoreactive (GLU-LIR) neurons in the adult and developing nervous system of Lymnaea. Altogether, 50–60 GLU-LIR neurons are present in the adult central nervous system. GLU-LIR labeling is shown in the interganglionic bundle system and at the varicosities in neuropil of the central ganglia. In the periphery, the foot, lip, and tentacle contain numerous GLU-LIR bipolar sensory neurons. In the juvenile Lymnaea, GLU-LIR elements at the periphery display a pattern of distribution similar to that seen in adults, whereas labeled neurons increase in number in the different ganglia of the central nervous system from juvenile stage P1 up to adulthood. During embryogenesis, GLU-LIR innervation can be detected first at the 50% stage of embryonic development (the E50% stage) in the neuropil of the cerebral and pedal ganglia, followed by the emergence of labeled pedal nerve roots at the E75% stage. Before hatching, at the E90% stage, a few GLU-LIR sensory cells can be found in the caudal foot region. Our findings indicate a wide range of occurrence and a broad role for glutamate in the gastropod nervous system; hence they provide a basis for future studies on glutamatergic events in networks underlying different behaviors.

Abbreviations: CNS, central nervous system • GLU, glutamate • GLU-L, glutamate-like • GLU-LIR, glutamate-like immunoreactive • NMDA, N-methyl-D-aspartate • PNS, peripheral nervous system


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Literature Cited
 
Glutamate (GLU) is a major excitatory neurotransmitter, widely distributed throughout the animal kingdom in both vertebrates and invertebrates. Its role is best characterized in different brain regions of higher vertebrates (Collingridge and Lester, 1989; Dingledine et al., 1999) and especially in neurons such as the pyramidal and granule cells of the cerebral and cerebellar cortex (Bliss and Collingridge, 1993; Freund and Buzsaki, 1996).

GLU as a neurotransmitter has been also studied in detail in invertebrates, mainly in arthropods (for an extensive review see, e.g., Walker et al., 1996). In both crayfish and insects, GLU is a principal excitatory transmitter in the peripheral nervous system (PNS) at neuromuscular contacts, and physiological and pharmacological experiments showed the presence of ionotropic (N-methyl-D-aspartate [NMDA] and kainate) glutamate receptors (Usherwood, 1994; Shupliakov et al., 1995; Walker, 1996; Aonuma et al., 1998; Ha et al., 2006). The distribution of GLU-like immunoreactive (GLU-LIR) neurons and the ultrastructural characterization of GLU-LIR synaptic varicosities in both the central nervous system (CNS; brain and segmental ganglia) and PNS (neuromuscular contacts) have been described in a number of arthropod species (Bicker et al., 1988; Watson, 1988; Nagayama et al., 2004). The organization of the GLU-LIR system in the different compartments of the cricket mushroom bodies has also been described (Schürmann et al., 2000).

GLU as a signal molecule also seems to be widely distributed and to function in gastropods and cephalopods. Early biochemical and anatomical studies using radiolabeled GLU indicated that the Helix brain contains a high level of this amino acid (Osborne et al., 1971). GLU was shown to act as an excitatory neurotransmitter at the second-order giant synapse of squid, and at neuromuscular contacts in the chromatophores of different cephalopod species (Messenger, 1996). Several neurons in Aplysia were found to use GLU as a fast excitatory neurotransmitter (Dale and Kandel, 1993; Fox and Lloyd, 1999, Klein et al., 2000). A role was attributed to GLU in feeding behavior (Quinlan et al., 1995; Jones et al., 1997) and neuronal sprouting (Bulloch and Ridgeway, 1989) in Helisoma. In the Lymnaea CNS a GLUergic interneuron was identified as a member of the feeding network in the buccal ganglion (Brierley et al., 1997).

The GLU-sensitive neurons were mapped in the Lymnaea CNS after a detailed pharmacological analysis (Nesic et al., 1996). GLU-sensitive neurons were also identified in the Planorbarius and Helisoma CNS (Bolshakov et al., 1991; Quinlan and Murphy, 1991). Most of the GLU-sensitive neurons proved to be cells that lacked NMDA receptors, whereas only sporadic data indicated the occurrence of NMDA (Moroz et al., 1993) and other metabotropic receptors (Walker, 1996). In the olfactory center (the procerebrum) of Limax, bursting neurons possess a chloride-channel-coupled GLU-receptor that seems to be involved in the propagation of oscillatory activities (Watanabe et al., 2003). Sensory-motor transmission in Aplysia is mediated through the activation of GLU receptors (Trudeau and Castellucci, 1993). A putative GLU receptor subunit was cloned from the Lymnaea CNS and was partially identical to the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-selective receptor subunit in the rat (Darlison et al., 1993).

At the same time, there is very little information on the visualization of GLU-containing neurons in the molluscan nervous system. Localization of GLU and GLU-transporters was shown in identified sensory neurons of the pleural ganglia of Aplysia (Levenson et al., 2000). In Lymnaea, GLU-LIR fibers were recently demonstrated in the inferior cervical nerve and the head retractor muscle; however, labeled nerve cell bodies in the CNS were not found (Kononenko and Zhukov, 2005). Therefore, the aim of our present study was to describe the localization of GLU-LIR neurons in the CNS and PNS of Lymnaea stagnalis, including its developmental aspects during both embryonic and juvenile life. In this way, we hope to fill a gap in our knowledge related to the gastropod signaling system, to encourage further investigations aiming at the GLUergic transporter and receptor systems, and to support functional and behavioral studies that involve GLUergic processes in Lymnaea.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Literature Cited
 
Animals and staging
Embryos and adult and juvenile specimens of Lymnaea stagnalis L., a pond snail, were used for our experiments. Adult snails were collected in their natural habitat and maintained thereafter in aquaria supplied with fresh Balaton water. Another population of snails originated from breeding stocks at the Free University, Amsterdam. A total of 25 adult snails were used. Egg masses and juveniles were collected from the laboratory population of adult snails. Embryonic development was staged on the basis of a specific set of morphometric, morphological, and behavioral features (Morill, 1982; Mescheriakov, 1990), and the stages were expressed as a percentage of total embryonic development (E0 [0%] to E100 [100%], Marois and Croll, 1992). Juvenile snails were categorized according to shell size (Croll and Chiasson, 1989). For our investigations, we used embryos of E50% to E90% (a minimum of 40 per stage) and juveniles of stages P1 to P4 (15–20 per stage).

Fixation
Preparations were fixed in 4% paraformaldehyde diluted in 0.1 mol l–1 phosphate buffer (PB). Embryos and P1–P2 juveniles were removed from the shell and fixed as whole-mounts. The CNS (including the circumesophageal ganglion complex and the pair of buccal ganglia) and peripheral tissue pieces (head region with tentacles and lips, lateral foot, heart, and salivary gland) were quickly dissected from older juveniles and adult snails, pinned out in Sylgard-coated petri dishes, and then covered with fixative. After fixation for 4 h or overnight at 4 °C, the preparations were washed thoroughly in PB and phosphate-buffered saline (PBS) containing 0.25% Triton X-100 (PBS-TX), and then processed for immunohistochemistry.

Immunohistochemistry
Immunohistochemical labeling was performed either on 14–16-µm serial cryostat sections cut horizontally from embryos and P1–P2 juveniles and placed on chromalum-gelatin-coated slides, or on 50–60-µm serial Vibratome slices taken horizontally from the dissected CNS and peripheral tissue samples of P3–P4 juveniles and adults. The preparations were incubated for 24–48 h at 4 °C in a polyclonal anti-GLU antiserum raised in rabbit (Sigma, St. Louis, MO) diluted 1:1000 in PBS-Triton-X 100 containing 0.25% bovine serum albumin (PBS-TX-BSA). After washing in several changes of PBS-TX, immunofluorescence was visualized by incubation for 16 h at 4 °C with a secondary IgG tagged with rhodamine (TRITC; DAKO, Glostrup, Denmark) diluted 1:50 in PBS-TX-BSA. After several rinses in PBS, the preparations were mounted in a 3:1 mixture of glycerol-PBS.

Preparations were viewed and photographed either on a Zeiss Axioplan compound microscope equipped with an appropriate filter set, using a Canon digital camera; or with an Olympus FV-300 laser scanning confocal microscope, in which the optical sections at consecutive images of 1–2 µm were viewed and saved. Figures were processed digitally with Corel Draw 12 software.

Control experiments
Three types of control experiments were performed as follows: (i) method control in which the primary anti-GLU antiserum was replaced by pre-immune normal rabbit serum; (ii) negative control in which the primary anti-GLU antiserum was simply omitted from the incubation medium; and (iii) preabsorption control, in the course of which the primary anti-GLU antiserum (diluted 1:1000) was incubated with 10, 20, or 100 µmol l–1 GLU-glutaraldehyde-BSA complex for 24 h at 4 °C, prior to being applied to the cryostat sections. Immunostaining was totally abolished following preabsorption experiments (see Fig. 5), and no immunostaining was observed in either the method- or negative-control preparations.


Figure 5
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  		Figure 5. Control panel showing cryostat sections taken from adult Lymnaea CNS (A, B) and juveniles of different developmental stages (C, P4; E, P3; D & F, P1/2) and stained for GLU-immunofluorescence labeling, following preabsorption with different concentrations (10 µmol l–1, 20 µmol l–1, and 100 µmol l–1 ) GLU-glutaraldehyde-BSA complex. No immunoreactivity can be observed in either the central ganglia or at the periphery. CG, cerebral ganglion; cpe, cerebro-pedal connective; db, dorsal body; es, esophagus; PaG, parietal ganglion; PeG, pedal ganglion; PlG, pleural ganglion; plpe, pleuro-pedal connective; r, right; ra, radula; st, statocyst; VG, visceral ganglion; asterisks, lateral foot. Scale bars = 160 µm (A); 80 µm (B); 100 µm (C); 40 µm (D); 240 µm (E); 100 µm (F).

 

   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Literature Cited
 
Localization of GLU-L immunoreactivity in the central nervous system of adult Lymnaea stagnalis
Application of anti-GLU antiserum resulted in specific immunolabeling of a relatively small set of nerve cells and a widely distributed dense network of fiber systems in both the ganglionic neuropils and the interganglionic structures (connectives and commissures) in the CNS of Lymnaea stagnalis. An interganglionic bundle system consisting of numerous GLU-LIR fibers and running through the circumpharyngeal ganglionic ring was also a characteristic feature of the organization of the GLU-LIR system. Of the 50–60 GLU-LIR neurons that could be found in the CNS, most occurred in the cerebral, pedal, and buccal ganglia and were organized in smaller cell groups. Only individual neurons displaying GLU-L immunoreactivity were seen in the rest of the CNS, and the two pleural ganglia were free of labeled cells. The GLU-LIR neurons were small (20–30 µm) or medium-sized (40–60 µm), and no large or giant neurons revealed immunoreactivity. GLU-LIR neurons were found on the dorsal-dorsomedial region of the ganglia. No labeled neurons were seen on the ventral surface. Most of the peripheral nerve trunks contained numerous immunostained fibers, whereas no traces of labeled processes were seen in the connective tissue sheath surrounding the ganglia and peripheral nerve trunks.

Cerebral ganglia.
In the cerebral ganglia, GLU-L immunoreactivity was detected in two symmetrical cell clusters, each consisting of 8–10 neurons. One of them was located in the anterior lobe (Figs. 1A, B and 6A) and another at the posterio-lateral edge of the ganglion, near the dorsal lobe (Fig. 6A). The two clusters appeared at different times of ontogenesis. The anterior lobe cell group could be speculated to be a part of the CB cluster because of the localization and size (about 20 µm in diameter) of the cells (Croll and Chiasson, 1989; Hatakeyama and Ito, 1999). GLU-LIR, represented by intensely fluorescing varicose fibers, was also detected in the neuropil (Fig. 1C). The cerebro-pedal, cerebro-pleural, and cerebro-buccal connectives, as well as the cerebral commissure were supplied by numerous GLU-LIR processes, indicating a well-developed interganglionic GLUergic connectivity. The lip nerves also contained a rich GLU-LIR innervation.


Figure 1
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  		Figure 1. GLU-LIR neurons in the CNS of adult and juvenile Lymnaea. Vibratome-slices, unless otherwise indicated. TRITC labeling. (A, B) GLU-LIR neurons (arrowheads) located in the anterior lobe of the cerebral ganglion. Note labeled axons in the neuropil (asterisks), cerebral commissure (cc), and cerebro-pedal connective (cpc). (C) Higher magnification detail of the neuropil of the cerebral ganglion, showing GLU-LIR varicose fibers (arrows). Cryostat section. (D) GLU-LIR neurons (arrowheads) located in the left buccal ganglion. bc, buccal commissure; asterisk, neuropil. Cryostat section. (E, F) Unipolar neurons (arrowheads) displaying immunoreactivity in the pedal ganglia of adult (E) and P4 juvenile (F) snail. Note initial axon segments (open arrowheads in E) projecting toward the neuropil. Asterisk (F), neuropil; arrow (F), pedal commissure. (G) Two labeled neurons (arrowheads) in the adult right parietal ganglion, showing also numerous labeled fibers in the neuropil (asterisks). irpa, internal right pallial nerve. Scale bars = 80 µm (A, B, D, F, G); 40 µm (C, E).

 

Figure 6
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  		Figure 6. Schematic representation of the distribution of GLU-LIR neurons in CNS of adult (A), P3–P4 juvenile (B), and P1–P2 juvenile (C) Lymnaea. Dorsal views, except cerebral ganglia shown in ventromedial view. The cerebral ganglia are shown with the commissures cut and folded out. Open and filled squares indicate cell clusters that started to display GLU-L immunoreactivity at the adult and the P3–4 stage, respectively. BG, buccal ganglion; CG, cerebral ganglion; PeG, pedal ganglion; PlG, pleural ganglion; PaG, parietal ganglion; VG, visceral ganglion. Nerves (unless otherwise stated): 1 postbuccal, 2 buccal commissure, 3 dorsobuccal, 4 laterobuccal, 5 ventrobuccal, 6 cerebro-buccal connective, 7 superior lip, 8 median lip, 9 tentacular, 10 cerebral commissure, 11 cerebro-pedal connective, 12 superior medial, 13 median pedal, 14 inferior pedal, 15 pedal commissure, 16 cerebro-pleural connective, 17 left parietal, 18 cutaneous, 19 anal, 20 intestinal, 21 genital, 22 right parietal. (D) Schematic drawing of major GLU-LIR peripheral nerve bundles in Lymnaea, based on combining observations obtained in both adult and juvenile preparations. GLU-LIR was detected in the lip nerves, optic nerve, tentacle nerve, the nerve bundle surrounding the buccal mass, and pedal nerves.

 
Buccal ganglia.
In the buccal ganglia, a symmetrical pair of GLU-LIR cell clusters, each consisting of 4–6 neurons of 20–30 µm, were located in a caudo-medial position near the cerebro-buccal commissure (Figs. 1D and 6A). In a small number of preparations, additional labeled cells could occasionally be observed in the vicinity of this group; these labeled cells were in a caudo-lateral position, near to the origin of the buccal (pharyngeal) nerves. The neuropil of the buccal ganglia was characterized by intensive labeling, containing throughout densely arranged varicose fibers (Fig. 1D). GLU-LIR fibers were also found in the buccal commissure (Fig. 1D), the cerebro-buccal connectives, and the different pharyngeal nerves. A unique feature of the immunolabeling characterized the buccal ganglia: intensely fluorescing varicose fibers were seen surrounding a number of neuronal cell bodies as well as projecting to the surface of the ganglia, where these fibers ran along the ganglion surface within the inner thin connective tissue.

Pedal ganglia.
Although the neuropil of the pedal ganglia displayed the most intensive GLU-LIR innervation of any ganglia of the CNS, only one group of GLU-LIR neurons was found: this group was located at the posterior margin of the ganglia, between the origin of the pedal-pleural connective and the inferior pedal nerve and the pedal commissure (Figs. 1E, F and 6A). This group usually contained 8–10 immunolabeled neurons, although the number of labeled cells was occasionally larger—as much as double. No individual GLU-LIR neurons could be demonstrated, not even any of the previously physiologically or neurochemically identified cells, such as the RPeD1 giant dopaminergic or the LPeD1 and PeV4 PeV5 giant serotonergic cells (Cottrell et al., 1979; Croll and Chiasson, 1989). Neither does the localization of the GLU-LIR cell group correspond to that of any other group of neurons previously described as displaying 5-HT, dopamine, or histamine immunoreactivity in the adult Lymnaea CNS (Kemenes et al., 1989; Elekes et al., 1991; Hatakeyama and Ito, 1999; Hegedüs et al., 2004). Strongly reactive varicose fibers were detected in the neuropil of the pedal ganglia, as well as in the pedal commissures (Fig. 1F); all pedal nerve trunks also contained labeled processes.

Viscero-parietal-pleural ganglion complex.
A total of 8–10 GLU-LIR neurons were found in this part of the adult Lymnaea CNS, occurring as individual cells in the visceral and parietal ganglia. The pleural ganglia did not contain labeled nerve cells. In the right parietal ganglion, four medium-sized (40–50 µm) neurons exhibited GLU-L immunoreactivity; these neurons were located in the center of the perikaryonal layer of the ganglion, as well as near the origin of the parietal-pleural connective or the internal right parietal nerve (Figs. 1G and 6A). One of the smaller cells located at the parietal-pleural connective could already be detected at the P4 juvenile stage (Fig. 6B). In the left parietal ganglion, a single large (ca. 70–80 µm) neuron and two small (20–30 µm) cells that displayed GLU-L immunoreactivity only when they reached adulthood were observed (Fig. 6). In the visceral ganglion of the adult snails, a single GLU-LIR neuron occurred in the center of the ganglion (Fig. 6). In spite of the small number of GLU-LIR neurons observed, all ganglia of the complex were densely innervated by immunolabeled varicose fibers (Fig. 1G). In addition, a reactive bundle system connected the different ganglion units of the complex, and also including the pair of the pedal ganglia, ran through the neuropil and connectives. This bundle system, like others observed after immunohistochemistry was applied to visualize different aminergic (histamine, Hegedüs et al., 2004) or peptidergic (Mytilus inhibitory peptide, Elekes et al., 2000; leukokinin I, Elekes et al., 1994) systems, seems to delineate a kind of GLU-LIR connectivity among the different units of the CNS, suggesting the presence of projection interneurons and a role for GLU (like other signaling molecules) in synchronizing network activities.

Localization of GLU-L immunoreactivity in the peripheral tissues of adult Lymnaea
GLU-LIR elements were detected in different peripheral tissue of the adult Lymnaea, such as the buccal mass, lip, foot, body wall, tentacle, mantle, and eye, whereas other tissues such as the heart and the salivary gland did not display GLU-LIR innervation. Two types, afferent (sensory) and efferent, of GLU-LIR innervation could be distinguished in the periphery. GLU-LIR sensory cells revealed the typical bipolar anatomy of sensory neurons with apical, sometimes robust, dendrites projecting toward the surface and thin efferent axons projecting toward the deeper regions of the peripheral organ (Fig. 2A–D, F). Labeled sensory neurons were distributed evenly along longer surface segments of the peripheral organ, lining up in high number near each other (see, e.g., Fig. 3C, F) rather than forming cell groups. Afferent GLU-LIR axons were collected in bundles entering the different peripheral nerves. The organization of the GLU-LIR peripheral system in adult Lymnaea was identical with that seen in the juvenile of the species during postembryonic development. GLU-L immunoreactivity was observed in the nerve bundles running toward or from the peripheral organs such as lip (Figs. 3C and 6D), foot (Figs. 3B, D, E, and 6D), and tentacles (Figs. 2E and 6D). The size and morphology of the sensory neurons observed differed somewhat from one peripheral organ to another. The diameter of the cell bodies of the sensory neurons in the lip was 15–30 µm, and the length of the apical dendrites reached about 30–40 µm (Fig. 2A–D). The lip and the antero-lateral foot were especially richly supplied with GLU-LIR sensory elements (Fig. 2C, D, F). The bipolar sensory neurons seen in the tentacle appeared as delicate, fine cellular components of the peripheral GLU-LIR system (Fig. 2E), composed of a small elongated cell body and long (about 50 µm) apical dendrites running to the surface (Fig. 2E). GLU-L immunoreactivity was also detected in the optic nerve and along the base of the eye (Figs. 2G and 6D).


Figure 2
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  		Figure 2. GLU-LIR neurons in the periphery of adult and P4 juvenile Lymnaea. TRITC labeling. (A, B, C, D) GLU-LIR sensory neurons (arrows) are present in high number along the edge of the lip (A, B) and the antero-lateral foot (C, D) of adult Lymnaea. Vibratome slices. Arrowheads in B and D indicate sensory dendrites. (E, F) GLU-LIR bipolar sensory neurons (arrowheads) located at the base of the tentacle (E) and in the anterior foot (F). Open arrowheads, sensory axons; es, esophagus. Cryostat sections. (G) The optic nerve (arrowhead) and the retina (arrow) also appear to display GLU-L immunoreactivity. Cryostat section. Scale bars = 80 µm (A, C, E, G); 40 µm (B, D, F).

 

Figure 3
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  		Figure 3. Early development (P1–P2 juvenile stages) of the GLU-LIR system in Lymnaea. Cryostat sections, TRITC labeling. (A, B) Horizontal views of embryos, showing labeled CNS (arrows) with ganglia (left cerebral, pleural, and parietal in A, and the pair of pedal ganglia in B) and connectives, as well as peripheral innervation (arrowheads) in the tentacles and lateral and caudal foot regions. Pedal nerves extending from the pedal ganglion (double arrowheads in B) to the caudal foot region display also intensive GLU-L immunolabeling. (C) Organization of the GLU-LIR sensory system in the lip-cerebral ganglion axis. Open arrowheads, sensory dendrites; arrowheads, lip nerve bundles; CG, cerebral ganglion. (D, E) GLU-LIR sensory elements (arrowheads) in the caudal foot. (F, G) Efferent GLU-LIR innervation of the buccal mass (F) and the caudal foot (G). F is a higher magnification detail of A, showing fine varicose fibers (arrowheads) arborizing in the buccal mass. In G, a dense network of varicose processes (arrowheads) is seen at higher magnification, taken from a non-surface, deeper region of the foot. Scale bars = 200 µm (A, B); 160 µm (C); 80 µm (D); 40 µm (E, F, G).

 
The buccal mass and deeper, non-surface, regions of the foot revealed efferent innervation by GLU-LIR varicose fibers, similar to that shown in developing juveniles (Fig. 3F, G), whereas the mantle was supplied by a network of intensely fluorescing, partly varicose fiber systems (not shown). The nerve bundle surrounding the buccal mass was also GLU-LIR (Fig. 6D).

Localization of GLU-L immunoreactivity during the development of Lymnaea
Postembryogenesis (juvenile stages).
The postembryonic development of the GLU-LIR system in Lymnaea showed some differences at the central and peripheral levels. In the CNS, the number of GLU-LIR neurons gradually increased (Fig. 6) and the ganglion neuropils and connectives/commissures were characterized by an intensive GLU-LIR innervation from the early juvenile stages (Fig. 3A–C). In contrast, the different peripheries (foot, lip, tentacle) already displayed an adult-like GLUergic innervation pattern from early (P1, 2) postembryonic development (Figs. 3A–G). The fully developed presence of the peripheral GLU-LIR system might explain the intensive immunoreactivity in the ganglion neuropils, suggesting its origin from the many afferent axons entering the ganglia. From early (P1, 2) juvenile stages, GLU-LIR neurons forming a pair of cell groups could be clearly distinguished in the pedal ganglia (Fig. 6C), corresponding to those seen in older juveniles (Fig. 1F) and adults (Figs. 1E and 6A, B). By the P4 juvenile stage the development of the GLU-LIR system in the cerebral ganglia continued with the appearance of the CB cell cluster in the anterior lobe, whereas the posterio-lateral cell group appeared later, by the P5 juvenile stage (Fig. 6B). Three individual medium-sized neurons were also added, two of them in the visceral ganglion and one in the right parietal ganglion (Fig. 6B). The total number of GLU-LIR neurons was about 30 at the P4 juvenile stage, which was half of that counted in the adult CNS. The rest of the whole population of the GLU-LIR neurons demonstrated in the adult Lymnaea CNS appeared in the cerebral and parietal ganglia by the end of juvenile life, but those in the buccal ganglia could be detected only by the beginning of adulthood (Fig. 6). Only one transient GLU-LIR neuron was found, which appeared in the visceral ganglion of the P4 juvenile and ceased to exhibit immunoreactivity when adulthood was reached (Fig. 6A, B).

The development of the GLU-LIR sensory system by the beginning of juvenile life was characterized by a dramatic increase in the number of sensory neurons around the entire foot and in the lip and tentacle regions (for comparison, see Fig. 3A, D–F vs. Fig. 4A, C, D); this is considered to be the most impressive event in the development of the GLUergic system in Lymnaea.


Figure 4
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  		Figure 4. GLU-LIR elements during the embryogenesis of Lymnaea. Cryostat sections, TRITC labeling. (A, B) Horizontal sections taken from whole embryos of stage E90%, one day before hatching. GLU-L immunoreactivity is detected in different ganglia of the CNS, including the pedal (arrowheads), cerebral (double arrowheads), right pleural (small arrow in B), and right parietal (arrow in B) ganglia, commissures, and peripheral nerve trunks. es, esophagus. (C) Enlarged view of the caudal foot region framed with dotted line in A, GLU-LIR showing axons of developing sensory cells (arrowheads). (D) Embryonic stage E75%, post-metamorphic, adult-like stage. Low magnification horizontal view of the ventral (foot) surface, containing a few labeled (cross-sectioned) axon bundle elements of pedal nerve trunks (arrowheads) and possible sensory axons at the periphery (open arrowheads). m, mouth. Scale bars = 200 µm (A, B, D); 80 µm (C).

 
Intensive GLU-L immunoreactivity was exhibited by putative sensory neurons in tentacles (Fig. 3A), lateral foot (Fig. 3A), and lip (Fig. 3C). The afferents originating from the sensory cells located in different peripheral regions, such as the caudal and lateral foot (Fig. 3D, E) and lip (Fig. 3C), were collected in axon bundles and forwarded in the form of larger bundles to the CNS (Fig. 3B, C). On the basis of these observations, the general innervation pattern of the GLU-LIR sensory system in the Lymnaea periphery could be reconstructed (Fig. 6D). The size of the sensory cells also increased significantly during postembryonic development. For example, in the foot, their longer diameter doubled during this time; in the lateral foot, the increase was even greater—from 8–11 µm (P3–P4 juveniles, Fig. 2F) to 20–34 µm (adults, Fig. 2D). In addition, GLU-LIR varicose fibers innervating the muscle bundles of the buccal mass (Fig. 3F) and the deeper muscular regions of the caudal foot (Fig. 3G) were also present from the P1 juvenile stage.

Embryonic development.
At the late E90% embryonic stage, strong GLU-L immunoreactivity characterized the neuropil of the ganglia of the circumpharyngeal ganglion ring as well as of the buccal ganglia (Fig. 4A, B). As in the early juvenile stages (P1, 2), no labeled cell bodies could be detected, perhaps owing to the rather strong background masking. However, the intensive immunolabeling of the commissural systems emerged from this background (Fig. 4B), and the connectives and peripheral bundles running to and from the foot, tentacular, and lip regions also appeared as intensely immunolabeled structures (Fig. 4A, B). In spite of the strong immunoreactivity in the CNS and peripheral nerve bundles, only a few GLU-LIR sensory elements could be found in the different peripheral regions, even late in embryonic development (E90%). Only in the caudal foot region could thin labeled fibers resembling sensory axons be seen (Fig. 4C). These fibers possibly belonged to developing sensory cell bodies, which might be absent or very low in GLU content along the surface of the tail. In the postmetamorphic E75% embryonic stage, the GLU-LIR system was strongly reduced compared to that in the E90% stage. At this time of embryogenesis, the immunolabeled central elements were represented by the pair of pedal ganglia, displaying GLU-L immunoreactivity in the neuropil. In addition, axon bundle elements corresponding to the pedal nerve trunks were immunopositive, as were a few thin fibers in the lateral and caudal foot (Fig. 4D).


   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Literature Cited
 
Our immunohistochemical findings clearly show that GLU is present in different neuronal elements of the central and peripheral nervous system of the adult and developing (embryonic and juvenile) pond snail Lymnaea stagnalis. The results also indicate that only a small population (50–60) of neurons displays GLU-L immunoreactivity in the adult CNS, whereas labeled sensory elements are widely distributed at certain peripheral regions (foot, lip, body wall, and tentacle), and the efferent (motor) innervation of muscular organs (buccal mass, foot) also includes putative GLUergic elements. This kind of differential distribution of central and peripheral GLU-LIR neurons suggests that GLU can be considered a transmitter candidate in the nervous system of the gastropod Lymnaea. The relatively small number of GLU-LIR neurons, combined with rich arborization and projection patterns at both central (ganglion neuropil, connectives and commissures) and peripheral (nerves and tissue) levels, indicate a broad involvement of this amino acid in different regulatory processes, including intra- and interganglionic integration, as well as afferent and efferent roles. As far as we know, this is the first study reporting on the distribution of the entire population of neurons that may contain (and function with) GLU in gastropods, thus providing an anatomical basis for the analysis of the role of GLU as a signal molecule used in the underlying behavior of neural networks.

Our results also show that the distribution of GLU-L immunoreactivity changes throughout embryonic development and during the first half (P1–P4) of postembryonic (juvenile) development, suggesting that the functional maturation of the GLUergic system is finished when the animal reaches adulthood. GLU seems not only to function as a transmitter in the Lymnaea nervous system, but also to be involved in the late development of certain cellular events necessary for adequate adult behavior.

Specificity and localization of the immunolabeling
We used a commercial (Sigma) anti-glutamate polyclonal antibody raised in rabbit to investigate the distribution of GLU-L immunoreactivity in both the adult and developing Lymnaea nervous system. The indirect immunofluorescence technique applied to both cryostat and Vibratome sections yielded an unequivocal labeling. That is, GLU-L immunoreactivity could be observed in cell bodies and corresponding axon (and dendrite) projections, in many cases with high resolution of varicose processes. We could observe a well-delineated differential distribution of the GLU-L immunoreactivity in a small number of neurons in the CNS and immunolabeling of peripheral sensory and efferent structures. However, a negative staining was observed in most of the central neurons and certain peripheral tissues tested (heart, salivary gland). Therefore, it is suggested that the anti-GLU antibody used in our study specifically labels elements containing GLU with great probability. In addition to the negative-control and method-control experiments, we also performed the specificity (preabsorption) test with the GLU-glutaraldehyde antigen complex. According to the specificity test, the GLU-L immunolabeling was completely abolished in the CNS and peripheral tissues of both adult and developing individuals of Lymnaea (Fig. 5). It indicates that the immunostaining observed represents intracellular GLU of the reactive neural components.

In an immunocytochemical study on sensory neurons located in the pleuro-pedal ganglia of Aplysia (Levenson et al., 2000), all neuronal somata displayed immunoreactivity, but its intensity was considerably higher in GLU-LIR sensory cells than in the rest of the neuronal population. This observation seems only partly in accord with our present results in Lymnaea, because in our case a good number of central neurons in the circumpharyngeal ganglion ring remained unstained, showing little or no background fluorescence (see, e.g., Fig. 1B, D–F).

GLU is a putative transmitter in the Lymnaea central nervous system
The localization of GLU-L immunoreactivity in a relatively small set of small and medium-sized neurons and dense varicose fiber systems in the Lymnaea CNS suggests that GLU is a transmitter candidate involved in neuronal interactions at both intra- and interganglionic levels. A possible transmitter role for GLU in Lymnaea CNS was indicated by the presence of NMDA receptors in the light yellow cells (Moroz et al., 1993). A putative GLU receptor subunit (LymGluR) was cloned from Lymnaea, displaying a partial homology with rat GLU receptor subunits (Darlison et al., 1993). In a detailed pharmacological study, Nesic et al. (1996) mapped the distribution of GLU-sensitive nerve cells in the different ganglia of Lymnaea. By comparing their mapping with the localization of GLU-LIR neurons visualized in our study, we see that the two systems do not overlap. Whereas a number of previously identified neurons responded to GLU application, none of them displayed GLU-L immunoreactivity in the adults or juveniles of our study. This clear distinction between the distribution of GLU-LIR and GLU-sensitive nerve cells in the Lymnaea CNS can be interpreted as indirect evidence for the specificity of the immunostaining obtained in our study, supporting the view that the GLU-LIR nerve cells contain GLU. In the study by Nesic et al. (1996), the VD4 neuron, a cardiorespiratory interneuron located in the visceral ganglion of the Lymnaea CNS (Janse et al., 1985; Syed and Winlow, 1991; Skingsley et al., 1993), depolarized its postsynaptic cells, and this effect could be mimicked only by GLU. Hence it was proposed that GLU is the transmitter of the VD4 interneuron. According to our map, the visceral ganglion of adult Lymnaea contains a single GLU-LIR neuron, and we assume that it corresponds to the VD4 interneuron. In contrast, a previously identified GLUergic interneuron (N2v) of the feeding network of the Lymnaea buccal ganglion (Brierley et al., 1997) was immunonegative in our study. The GLU-like immunopositive cells were far fewer than the GLU-sensitive cells (Nesic et al., 1996). We suggest two possible explanations for this contradiction. (1) GLU-L immunoreactivity was detected not only in cell bodies but also in many varicosities throughout the CNS. Neurotransmitters can be released from varicosities, which are considered to function like the presynaptic terminals of nerve cells both in vertebrates (Tojima et al., 2000; Brain et al., 2001; and see Boehm and Kubista, 2002, for a review) and in invertebrates (Fox and Lloyd, 1999; Croll et al., 2004; Sykes and Condron, 2005). Although the number of GLU-LIR cells was actually small, axons originating from them seem to have many varicosities, thus innervating a good part of the CNS neuropil and representing the main components of GLUergic transmission in Lymnaea. (2) GLU-L immunoreactity was also observed in numerous putative sensory neurons located in different peripheral areas. Consequently, GLU may be mainly involved in peripheral neurotransmission, with a lesser role in central events.

Our preliminary biochemical measurements also support a transmitter role for this amino acid in the Lymnaea CNS, showing a specific one-component uptake system for 3H-GLU, which can be blocked pharmacologically, and the release of GLU after stimulation or in the presence of a high K+ concentration (Elekes, Mita, Hiripi, Hatakeyama, and Ito, unpubl. obs.). A wide regulatory role for GLU at the cellular membrane level was also demonstrated in the CNS of other gastropod species closely related to Lymnaea, such as Helisoma and Planorbis (Jones et al., 1987; Bolshakov et al., 1991; Quinlan et al., 1991), as indicated by a series of pharmacological-physiological experiments.

GLU appears to be involved in both afferent and efferent events in the Lymnaea peripheral nervous system
GLU-LIR neurons also appear to be important afferent and efferent components of signaling systems at the Lymnaea periphery. A great number of GLU-LIR sensory neurons were found in the oral (lip), tentacular, and pedal (foot) areas from early (P1, 2) juvenile stages. In Aplysia, Levenson et al. (2000) found that sensory neurons located centrally in the pleuro-pedal complex expressed GLU-L immunoreactivity. As for other amino acid transmitters, GABA ({gamma} aminobutyric acid)-L immunoreactivity has not been reported to occur in sensory neurons at the periphery of different gastropod species (Cooke and Gelperin, 1988; Richmond et al., 1991; Soinila and Mpitsos, 1991; Hernádi, 1994). A mixed situation is also characteristic for the presence and organization of the (mono)aminergic peripheral (sensory) systems studied in snails. Tyrosine hydroxylase (TH)-LIR sensory elements were observed in the lip, foot, and tentacular regions of Lymnaea (Croll et al., 1999), whereas histamine-LIR (Hegedüs et al., 2004) and 5-HT-LIR (McKenzie et al., 1998) sensory elements were not found at all.

Another peripheral organ seems to be the visual system of Lymnaea, in which GLU-LIR innervation was present. The optic nerve and—from early juvenile stages (P1–2)—a network of varicose fibers right beneath the retina displayed immunolabeling (Figs. 3D and 6), but none was found in the eye. Although Lymnaea has two types of photoreceptor cells in the eye (type A and type T cells; Sakakibara et al., 2005), we could not observe GLU-L immunoreactivity in them. Michel et al. (2000) previously showed strong GLU-L immunoreactivity in the photoreceptor cells, whereas the optic nerve was only faintly stained in the marine mollusc Bulla gouldiana. In the Lymnaea optical system, GLU may play a role in transmitting or modulating visual information toward the CNS, rather than being involved in the process of primary photoreception.

GLU-LIR efferent innervation by fine varicose fibers was also demonstrated in a few peripheral tissues, such as the foot and buccal musculature (Fig. 3F, G), but not in the heart and the salivary gland, suggesting that GLU also has a role in neuromuscular transmission. In a recent study on the innervation of the head retractor muscle of Lymnaea (Kononenko and Zhukov, 2005), GLU-LIR fibers were demonstrated in the corresponding peripheral nerve and along muscle fibers, but no immunolabeled neuronal cell bodies could be detected in the CNS. This contradiction between our finding and that of Kononenko and Zhukov might be explained by differences in methods: they used wholemount preparations of the Lymnaea CNS (and head retractor muscle), but we used either 50–60 µm Vibratome or 14–16-µm cryostat sections obtained from the circumpharyngeal ganglion ring. Our preparation allows better penetration of the antibody, especially to reach the smaller neurons in deeper levels of the ganglia. Furthermore, Kononenko and Zhukov used glutaraldehyde in the fixative, which can result in poorer penetration when labeling wholemount preparations.

GLU is the fast excitatory transmitter at buccal neuromuscular synapses in Aplysia (Fox and Lloyd, 1999). In cephalopods, GLU is an excitatory transmitter at neuromuscular contacts of several peripheral tissues, such as the chromatophores, mantle, and fin (Messenger, 1996). A number of previous studies demonstrated the role of GLUergic neuro-glandular synapses in the salivary cells of Helisoma (Quinlan and Murphy, 1991; Bahls et al., 1995; Quinlan et al., 1995). However, we failed to demonstrate immunolabeling either in any of the B1-4 motoneurons or in the salivary gland itself. GLU is also the principal excitatory neurotransmitter in motoneurons of arthropods (Johansen et al., 1989; Usherwood, 1994; Shupliakov et al., 1995, Burrows, 1996; Shayan et al., 2000).

Developmental aspects
Our findings are the first to demonstrate the presence of GLU-L immunoreactivity in the developing nervous system of a mollusc, suggesting a role for GLU in the neurogenesis and ontogenesis of a snail. In the present study, it was shown that the development of the GLU-LIR system is characterized by a gradual, but uneven, maturation, in the course of which labeled neuronal cell bodies were observed for the first time in the CNS of hatchlings/P1 juveniles, and GLU-LIR sensory neurons at the periphery also appeared in significant numbers only by this time of development. Efferent innervation of certain peripheral regions is also a typical part of the GLU-LIR system after hatching. In contrast, no cellular elements displaying GLU-L immunoreactivity could be found during embryogenesis, apart from a few sensory elements localized in the tail and a strong but partly undifferentiated immunoreactivity in the ganglion neuropils. An especially intriguing observation is the sudden appearance of numerous GLUergic sensory cells by the time of hatching (P1 juvenile stage) at certain rostral and caudal regions of the periphery, such as the lips, tentacle, lateral foot, and tail. It appears that GLU as a putative signaling molecule enters both peripheral and central regulatory process by the beginning of the postembryonic (juvenile) life. The intensive development of a GLU-LIR sensory system at the periphery after hatching—by the beginning of juvenile life—of Lymnaea can be compared to what we have found for the TH-LIR system (Croll et al., 1999); however, with a somewhat different distribution. In these two latter cases, the mantle was also richly supplied with labeled sensory neurons. This phenomenon is probably related to the fact that GLU is used in chemical and mechanical sensory processes necessary for behaviors that underlie the free-living foraging life that begins with hatching.

Comparing the time-scale of appearance and distribution of GLU-LIR neurons during embryogenesis with that of other transmitter systems studied in Lymnaea, such as serotonin (5-HT) (Marois and Croll, 1992; Voronezhskaya and Elekes, 1993), dopamine (DA) (catecholamines; Voronezhskaya et al., 1999), octopamine (OA) (Elekes et al., 1996), {gamma}-amminobutyric acid (GABA) (Hatakeyama and Ito, 2000), and nitric oxide (NO) (Serfözö et al., 1998, 2002), both similarities and differences can be established. The time-scale of the development of the GLU-LIR system resembles that of GABA (Hatakeyama and Ito, 2000), and partly that of OA (Elekes et al., 1996) and DA (Voronezhskaya et al., 1999). In the case of GABA, no immunoreactive elements could also be demonstrated in embryos but only in juvenile stages (Hatakeyama and Ito, 2000), whereas the first OA-LIR (Elekes et al., 1996) and tyrosin hydroxylase (TH)-LIR (dopaminergic; Voronezhskaya et al., 1999) neurons appeared by the late embryonic stage (E85%) in the Lymnaea CNS. In contrast, the embryogenesis of 5-HT-LIR neurons started by an early stage (E35%; Marois and Croll, 1992; Voronezhskaya and Elekes, 1993). Although no peripheral elements displayed immunolabeling for GABA and OA, a few GLU-LIR elements were present at late embryonic development, which was similar to what was observed for DA (Voronezhskaya et al., 1999). During the embryogenesis of the NOergic system in Lymnaea (Serfözö et al., 1998, 2002), only peripheral sensory cells in the epithelial layer of the upper (esophageal) alimentary tract displayed reactivity until hatching.


   Acknowledgments
 
Postdoctoral Fellowships for Research Abroad from the Japan Society for the Promotion of Science; Grant number: 577 (D.H.). Grant sponsor: The Japan Society for the Promotion of Science Grant; Grant numbers: 16370033 and 17657049 (E.I.). Grant sponsor: Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology, Scientific Research on Priority Areas (Area No. 454); Grant No: 17075001 (H.A.). Grant sponsor: Hungarian Scientific Research Fund (OTKA); Grant No. 49090 (K.E.). The skillful technical assistance of Ms. Zsuzsanna N. Fekete, and Mr. Boldizsár Balázs is greatly appreciated. Authors are grateful to Dr. Christopher J. H. Elliot (University of York, U.K.) for reading and editing the English.


   Footnotes
 
Received 22 February 2007; accepted 21 May 2007.

* Present address: Naturwissenschaftliche Fakultät III, FR 8.3—Biowissenschaften, Zoologie/Physiologie, Universität des Saarlandes, D-66041 Saarbrücken, Germany. Back


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